Chemokines are expressed in a myeloid cell-dependent fashion and mediate distinct functions in immune complex glomerulonephritis in rat.

Using anti-glomerular basement membrane nephritis in rats, we investigated the mechanisms underlying in situ chemokine expression and the in vivo function of these cytokines during the acute phase of this model. We observed that CXC chemokine expression was monophasic and paralleled neutrophil (PMN) influx, whereas CC chemokine expression was biphasic with peaks coinciding with the influx of PMNs and macrophages (Mphi). The initial peak of chemokine expression was attenuated by decomplementation, neutropenia, and leukopenia, while the latter peak was attenuated only by leukopenia and augmented in the accelerated form of this disease model, corresponding to an increase in Mphi influx. Differential expression of chemokines by PMNs and Mphi was not an intrinsic property of these cells, as these leukocytes expressed similar profiles of chemokines in vitro. Immunostaining for Mphi inflammatory protein-1alpha, a CC chemokine, in acute nephritis validated that expression during acute nephritis was accompanied by local protein production. Moreover, neutralizing Ab to Mphi inflammatory protein-1alpha attenuated the acute phase proteinuria, but not the accompanying influx of PMNs. Neutralizing Ab to cytokine-induced neutrophil chemoattractant (a CXC chemokine), in comparison, inhibited both PMN influx and proteinuria. A combination of both Abs was not significantly more effective than either alone. In sum, the influx of myeloid cells is necessary for local chemokine expression in anti-glomerular basement membrane nephritis, although the differential expression of CXC and CC chemokines must involve additional factors. CXC and CC chemokines also mediate distinct, but overlapping, pathophysiologic roles in the acute phase of this model.

GRO chemokines: a transduction, integration, and amplification mechanism in acute renal inflammation.

We recently observed that cytokine-induced neutrophil chemoattractant (CINC), a GRO chemokine, contributes to neutrophil migration into the inflamed glomerulus in rat. Therefore, we sought to clarify how expression of the GRO chemokines, CINC and macrophage inflammatory protein-2 (MIP-2), is regulated in mesangial cells in vitro and the kidney in vivo. Mesangial cells expressed both GRO chemokine mRNAs in response to mediators of acute renal inflammation [interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides (LPS)], but not chronic renal inflammation (transforming growth factor-beta 1), with CINC mRNA expression predominating over MIP-2. The kinetics of GRO chemokine mRNA expression in response to both IL-1 beta and TNF-alpha (but not LPS) paralleled those defined for polymorphonuclear leukocyte (PMN) migration during nephritis in vivo. IL-1 beta and TNF-alpha displayed nonparallel concentration-response relationships for GRO chemokine mRNA expression, and together were synergistic together rather than additive. Expression of GRO chemokine mRNAs in response to both cytokine agonists, however, was inhibited by genistein, a tyrosine kinase inhibitor. GRO chemokine mRNAs were rapidly expressed in inflamed glomeruli during immune complex glomerulonephritis with MIP-2 predominating over CINC. Expression of both chemokines was substantially inhibited by complement, leukocyte, and PMN depletion. In sum, GRO chemokines are expressed coordinately by mesangial cells and inflamed glomeruli and appear both to transduce the response to mediators of acute inflammation into a chemotactic signal and to amplify this response both temporally and quantitatively.(ABSTRACT TRUNCATED AT 250 WORDS)

Discordance between macrophage arachidonate metabolic phenotype and the expression of cytosolic phospholipase A2 and cyclooxygenase.

Macrophages (M phi s) exhibit variations in their ability to release and metabolize arachidonate (AA) depending on their state of activation, differentiation, and tissue origin. In order to understand these variations on a molecular level, we determined whether differences in AA release and metabolism by murine peritoneal M phi s could be explained in terms of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX) expression. Resident M phi s exhibited greater COX capacity (conversion of exogenous AA to PGE2) but lower phospholipase (PLase) activity (release of endogenous AA) than elicited M phi s. Activation of resident M phi s in vivo with endotoxin increased both their PLase activity and COX capacity. Despite the observed differences in PLase activity, peritoneal M phi s under all conditions expressed similar amounts of cPLA2 mRNA and protein. All M phi s exhibited COX-1 mRNA and protein (i.e., the constitutive isoform of COX), although elicited M phi s exhibited increased mRNA for COX-1 but decreased levels of protein, relative to resident M phi s. Elicited (but not resident) cells also exhibited COX-2 mRNA but not COX-2 protein (i.e., the inducible form of COX). Despite the increased COX capacity of resident cells with in vivo activation, their expression of COX-2 mRNA and protein was equivalent to that of unactivated cells, becoming apparent only after cell adherence in vitro. In sum, there is no simple relationship between the ability of M phi s to release and metabolize AA, and the expression of cPLA2 or COX isoforms. Moreover, adherence appears to be important for the expression of COX-2 by M phi s.

Distinct pattern of embryonic expression of the sea urchin CyI actin gene in Tripneustes gratilla.

Cloning and sequencing of Tripneustes gratilla genomic DNA and cDNA encoding a developmentally regulated, embryonic messenger RNA, referred to as Tg616, revealed an actin-encoding gene orthologous to the CyI actin gene described from Strongylocentrotus purpuratus. Tg616 and SpCyI share: (1) 150 nucleotides of highly conserved sequence 5' of the transcription start site, (2) 95% nucleotide sequence identity in the protein encoding regions, which specify identical amino acid residues in 375 of 377 positions, and (3) extensive nucleotide sequence identity in the 3' untranslated region of their messenger RNAs. Tg616 was therefore designated TgCyI. In situ hybridization shows sequential activation of TgCyI in various cells of the embryo. TgCyI mRNA becomes abundant in primary and secondary mesenchyme cells as they prepare to enter the blastocoel, in prospective aboral ectoderm cells at blastula stage, in gut cells during gut differentiation, and in oral ectoderm at pluteus stage. This pattern of embryonic gene expression is more complex than any of the major patterns of developmentally upregulated genes observed in S. purpuratus embryos and is distinct from SpCyI expression which is progressively restricted to the gut and oral ectoderm.

Regulation of cytokine-induced neutrophil chemoattractant (CINC) mRNA production in cultured rat cells.

Rat cytokine-induced neutrophil chemoattractant (CINC) is an 8-kD polypeptide originally purified from media conditioned by interleukin-1 beta (IL-1 beta)-stimulated 52E, an epithelioid clone derived from the normal rat kidney (NRK) cell line. Using a fibroblastic clone of NRK cells, 49F, we found that lipopolysaccharide (LPS), IL-1 beta, and tumor necrosis factor-alpha (TNF-alpha) each induce synthesis of CINC mRNA and CINC, although in qualitatively and quantitatively different patterns. Through deadenylation experiments and by probing with oligonucleotides, we discovered that the smaller of the two major CINC transcripts appears to arise from the larger as a result of poly(A) tail removal and/or 3' cleavage.

Investigation of possible autocrine functions for rat GRO/CINC (cytokine-induced neutrophil chemoattractant).

Rat cytokine-induced neutrophil chemoattractant (CINC) is an eight kilodalton polypeptide originally purified from media conditioned by interleukin-1 beta stimulated 52E, an epithelioid clone derived from normal rat kidney (NRK) cells. Using a fibroblastic clone of the NRK cells, 49F, we found expression of the CINC gene to be induced by either serum or cytokines in growth-arrested cultures within 1 hour of stimulation. There was no observable CINC expression in exponentially growing cells in the absence of cytokine stimulation. CINC protein had no significant effect on 3H-thymidine incorporation or growth rate of NRK49F. We have observed that CINC is constitutively produced by some transformed NRK cells, clone RC20, suggesting an association with the expression of a transformed phenotype. Unlike the parent 49F, RC20 cells are capable of growth in soft agar and serum-free media and form highly metastatic tumors in nude mice. We have examined the possible autocrine functions of CINC and its possible links to the expression of the transformed phenotype by these cells. The use of a blocking CINC polyclonal antibody demonstrated that CINC did not function as an autocrine growth factor for RC20. Though CINC is a potent chemoattractant for neutrophils, it did not induce migration of either RC20 or 49F cells. CINC only moderately promoted adhesion of RC20 cells when used as a matrix protein. These data do not support the hypothesis that production of CINC by the RC20 cells provides an obvious advantage for the transformed cells constitutively producing it.

High-level expression of cytokine-induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: purification and production of blocking antibodies.

Significant levels of cytokine-induced neutrophil chemoattractant (CINC) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC-50 0.5 nM). A fusion protein of glutathione-S-transferase and CINC (GST-CINC) was produced in E. coli. Anti-CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST-CINC affinity chromatography. Both goat and rabbit anti-CINC antibody preparations at 4 micrograms/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity-purified blocking anti-CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells.

A PCR procedure to determine the sequence of large polypeptides by rapid walking through a cDNA library.

A procedure that uses the PCR to make rapid successive steps through a random-primed cDNA library has been developed to provide a method for sequencing very long genes that are difficult to obtain as a single clone. In each successive step, the portions of partial clones that extend out from the region of known DNA sequence are amplified by two stages of PCR with nested, outward-directed primers designed approximately 50 bases in from the end of the known sequence, together with a general primer based on the sequence of the vector. This procedure has been used to determine the coding sequence of the cDNA for the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla. By starting from a single parent clone, whose translated amino acid sequence overlapped the microsequence of a tryptic peptide of the beta heavy chain, and making 3 such walk steps downstream and 14 walk steps upstream, we obtained a sequence of 13,799 base pairs that had an open reading frame of 13,398 base pairs. This sequence encodes a polypeptide with 4466 residues of Mr 511,804 that is believed to correspond to the complete beta heavy chain of ciliary outer arm dynein.

Effects of a variety of cytokines and inducing agents on vascular permeability factor mRNA levels in U937 cells.

Vascular permeability factor (VPF) is an approximately 40-kDa disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth and angiogenesis. Little is known about VPF gene regulation. In this study, we investigated the effects of a variety of cytokines and inducing agents on VPF mRNA levels in the monocyte-like U937 cell line. Transforming growth factor-beta 1 caused a 1.8-fold increase in VPF mRNA levels after 4 hours, followed by a decline to basal levels by 18 hours. Phorbol 12-myristate 13-acetate, a potent inducer of the differentiation of U937 cells, caused a 12.5-fold increase in VPF mRNA levels at 24 hours, coinciding with the differentiation of these monocyte-like cells into macrophage-like cells.

Characterization and expression of two sea urchin homeobox gene sequences.

We describe two homeobox sequences, TgHbox5 and TgHbox6, isolated from the Hawaiian sea urchin Tripneustes gratilla using a Drosophila Sex combs reduced probe. Sequence analysis shows that the encoded TgHbox5 homeodomain shares only 30-52% amino acid identity with homeodomains encoded by previously characterized genes, establishing that it is a divergent homeobox that is not in any known class of homeoboxes. TgHbox5 is expressed in the embryo as two major developmentally regulated transcripts. one at 5.0 kilobase (kb) appearing by blastula stage and the other at 2.7 kb appearing at pluteus stage. Multiple transcripts from TgHbox5 are present at a much lower level in adult tissues and are predominantly expressed in small and large intestines. The TgHbox6 homeobox is an Antenna-pedia-class homeobox, which appears not to be expressed during embryogenesis but produces abundant 3.6 and 3.2 kb transcripts in the six adult tissues examined.

Progressively restricted expression of a homeo box gene within the aboral ectoderm of developing sea urchin embryos.

A homeo box-containing gene, Hbox1 is expressed in an unusual and highly conserved spatial pattern in embryos of two different species of sea urchin, Tripneustes gratilla and Strongylocentrotus purpuratus. Hybridization in situ shows that this mRNA accumulates initially throughout the aboral ectoderm; however, between blastula and pluteus stages, the region containing Hbox1 mRNA retracts gradually until only a small area around the vertex is labeled in pluteus larvae. Aboral ectoderm appears cytologically uniform and also accumulates uniform levels of other tissue-specific mRNAs. Therefore, the Hbox1 pattern reveals a previously unsuspected heterogeneity of aboral ectoderm cells and a polarity within this tissue. In S. purpuratus, the Hbox1 gene product probably is not involved in initial specification of cell fate, as this message does not achieve a significant fraction of its peak abundance until almost hatching blastula stage, well after the time aboral ectoderm cells have initiated a tissue-specific program of gene expression. RNA blot and RNase protection analyses revealed low levels of Hbox1 mRNA in all adult tissues examined. However, this message was not detectable in mature eggs, suggesting that the Hbox1 gene does not have a maternal function. In addition to highly conserved spatial and temporal patterns of expression, the homeo box genes of these two urchin species also are conserved highly in sequences outside the homeo domain, despite the divergence of these two species (30-45 my). Two notable features of the protein shared with several vertebrate homeo proteins are a short conserved sequence encoded by an exon upstream of that encoding the homeo domain and a large region of high serine and proline content.

Stage- and tissue-specific expression of two homeo box genes in sea urchin embryos and adults.

We report the isolation of two different homeo box genes, HB3 and HB4, from the Hawaiian sea urchin Tripneustes gratilla. DNA sequencing revealed a definitive Antennapedia (Antp) class homeo box in each gene. Southern transfer hybridizations showed the genes to be single-copy. A 5.7-kb transcript of the HB3 gene was found in ovary, testis, small intestine and gastrula poly(A)+ RNA. The HB4 gene produces three transcripts. A 3.7-kb and a 4.4-kb transcript are expressed during embryogenesis. A 3.5-kb transcript appears in each of the adult tissues studied. The HB4 gene appears to be the sea urchin cognate of the Drosophila infrabdominal-7 (iab-7) gene, the mouse Hox 1.7 and Hox 3.2 genes and the Xenopus X1Hbox 6 gene. An examination of Antp class homeo box genes in deuterostomes indicates that a chromosomal duplication has taken place in the evolutionary line leading to the vertebrates after the divergence of the echinoderms. Thus, the sea urchin represents the primitive condition.

A gene expressed in the endoderm of the sea urchin embryo.

Using a previously cloned, developmentally regulated mRNA sequence expressed predominantly in the endoderm of sea urchin pluteus larvae, we isolated genomic clones and additional cDNA clones to define the gene and the protein it encodes. Nucleic acid sequencing revealed that the gene consists of four exons interrupted by three introns and spans approximately 3600 bp. It encodes a low-molecular-weight protein with polar ends. A stretch of Glu and Asp residues at its carboxyl terminus suggests that it is a nucleic acid-binding protein and a stretch of four Lys residues near the amino terminus suggests a nuclear localization signal.

An engrailed class homeo box gene in sea urchins.

The homeo box, a conserved DNA element first recognized in Drosophila development-controlling genes, is present in the genomes of many higher metazoan species and provides a valuable probe for the isolation of regulatory genes from diverse phylogenetic groups. We have employed these probes to isolate and study the homeo-box genes in sea urchins. As in other species, the sea urchin homeo boxes fall into at least two classes defined by nucleotide sequence similarity to the homeo boxes of the Drosophila Antennapedia (Antp) and engrailed (en) genes. In this study, we characterize the only detectable sea urchin en class homeo box. Its nucleotide sequence similarity and lack of an intron indicate that it is more closely related to the two mouse en class homeo boxes than to the two Drosophila en class homeo boxes. These relationships are most parsimoniously explained if the single sea urchin en class homeo-box gene represents the primitive condition and the two mouse and the two Drosophila en class homeo-box genes represent independent duplications which occurred in the evolutionary lines leading to the vertebrates and arthropods, respectively. The most abundant en class gene transcripts detected by gel transfer analysis of RNA extracted from sea urchin tissues were found in Aristotle's lantern. Rare transcripts were present in ovary, testis and coelomocytes.

Stage-specific expression of a homeo box-containing gene in the non-segmented sea urchin embryo.

Hybridization of Drosophila homeo box DNA probes to Southern transfers of genomic DNA from the Hawaiian sea urchin Tripneustes gratilla has revealed that the sea urchin genome contains at least five homeo boxes. Examination of the DNA from several individuals shows that the sequences flanking these homeo boxes exhibit little restriction fragment length polymorphism, indicating they are more highly conserved than the majority of sea urchin DNA. Several clones in a T. gratilla genomic DNA library which hybridized with Drosophila homeo box probes were identified, and one found to be transcribed during embryogenesis was selected for further study. Southern transfer hybridizations showed the cloned gene to be single-copy. DNA sequencing of the sea urchin gene defined a homeo box 70-73% homologous to the Drosophila homeo box probes and an encoded homeo domain 78-88% homologous to those encoded by the probes. Hybridization of DNA probes from the sea urchin homeo box-containing gene to Northern transfers of embryonic RNA demonstrated that the gene produces two transcripts of 6.9 kb and 7.7 kb. Transcripts first accumulate at blastula stage and increase to a maximum level at gastrula stage before decreasing considerably in abundance by pluteus stage.

Structural analysis of ribosomal RNA genes from the Hawaiian sea urchin species, Tripneustes gratilla.

Ribosomal gene sequences (rDNA) were isolated from the genomic DNA of the Hawaiian sea urchin Tripneustes gratilla by cloning in plasmid and phage vectors. The rRNA coding regions in four clones were localized by hybridizing Southern transfers of endonuclease-digested DNA with 32P-labeled 26S rRNA and 32P-labeled 18S rRNA. Three of the rDNA clones were isolated from a library of DNA from a single sea urchin and represent the two major types of rDNA repeats present in that individual's genome. Both repeat types appear identical within their rRNA coding regions but are dissimilar in an area of the nontranscribed spacer (NTS) adjacent to the 3' end of the 26S coding region. The cloned rDNA repeats were shorter than their genomic counterparts due to deletions occurring within internally repetitious NTS domains, probably as a result of unequal recombination during phage propagation.

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.